The invention relates to methods of genetically transforming plant plastids, and more specifically to genetically transforming the plastid genomes of Brassica plant species.
The plastids of higher plants are an attractive target for genetic engineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts, etioplasts, chromoplasts, etc.) are the major biosynthetic centers that, in addition to photosynthesis, are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. In general, plant cells contain 500-10,000 copies of a small 120-160 kilobase circular genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which potentially can result in very high levels of foreign gene expression. In addition, plastids of most plants are maternally inherited. Consequently, unlike heterologous genes expressed in the nucleus, heterologous genes expressed in plastids are not pollen disseminated, therefore, a trait introduced into a plant plastid will not be transmitted to wild-type relatives.
Plastids of higher plants present an attractive target for genetic engineering. As mentioned above, plastids of higher plants are maternally inherited. This offers an advantage for genetic engineering of plants for tolerance or resistance to natural or chemical conditions, such as herbicide tolerance, as these traits will not be transmitted to wild-type relatives. A review of plastid transformation of flowering plants is provided by Maliga (1993) Trends in Biotech. 11:101-107, the entirety of which is incorporated herein by reference.
Unfortunately, successful plastid transformation techniques described thusfar for higher plants have been limited to model crop plants such as tobacco (U.S. Pat. No. 5,451,513; Svab et. al. (1990), Proc. Natl. Acad. Sci. USA 87:8526-8530 and Svab et al. (1993), Proc. Natl. Acad. Sci. USA 90:913-197) and Arabidopsis (Sikdar, et al. (1998) Plant Cell Reports 18:20-24). Furthermore, the methods described for Arabidopsis plants, produce infertile regenerates. PCT Publication WO 97/32977 also describes methods for the plastid transformation of Arabidopsis and provides prophetic examples of plastid transformation of Brassica plastids. However, transplastomic Brassica plants have not been produced to date using the methods described therein. Thus, for practical applications of genetic engineering techniques to crop plant plastids, chloroplast transformation techniques for a wide variety of crop plants, such as Brassica species, are needed in the art.
The production of chloroplast transformation methods applicable to crop species other than tobacco and Arabidopsis is needed in the art. Such methods provide for a novel means of genetic engineering via plastid transformation to an attractive alternative to nuclear expression of agronomically as well as qualitatively important traits via genetic engineering of plant plastids.
Relevant Literature
Stable transformation of plastids has been reported in the green algae Chlamydomonas (Boynton et al. (1988) Science 240:1534-1538) and most recently in higher plants (Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917). These methods rely on particle gun delivery of DNA containing a selectable marker and targeting to the plastid genome by homologous recombination.
The complete DNA sequences of the plastid genomes from liverwort (Ohyama et al. (1986) Nature 322:572-574), rice (Hiratsuka et al. (1989) Mol. Gen. Genet. 217:185-194), and tobacco (Shinozaki et al. (1986) EMBO J. 5:2043-2049) have been reported.
The present invention provides constructs and methods for the transformation and regeneration of plants containing plant cells, the plastids of which have been stably transformed by a foreign DNA of interest. The method generally comprises transforming a Brassica plant cell plastid with a DNA construct; selecting for cells which contain the DNA construct; and obtaining a mature multicellular plant, the cells of which contain the DNA construct in the plant cell plastid.
The instant invention also provides methods for transforming the plastids of Brassica plant cells with a DNA construct generally comprising, in the 5xe2x80x2 to 3xe2x80x2 direction of transcription, a promoter region functional in a plant cell plastid, a DNA sequence of interest, and a transcription termination region functional in a plant cell plastid.
Furthermore, the present invention also provides the multicellular Brassica plant obtained by the methods described herein.
The invention also provides a multicellular Brassica plant, the plastids of which have been transformed with a DNA construct of interest.
The invention also provides a method for obtaining a Brassica plant cell, of which the plastid has been stably transformed with a DNA construct, comprising in the 5xe2x80x2 to 3xe2x80x2 direction of transcription, a promoter functional in a plant cell plastid, a DNA sequence encoding a green fluorescent protein (herein referred to as GFP), and a transcriptional termination region functional in a plant cell plastid.